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The Study of Microbial Structure: Microscopy and Specimen Preparation

1. Microscopy techniques allow observation of microscopic structures through use of lenses and light or electron beams. Specimen preparation through staining and fixing is required to visualize microscopic structures. 2. Various microscopes like bright-field, dark-field, phase-contrast, and fluorescence microscopes use different optical configurations and light sources to visualize specimens. Electron microscopes like TEM and SEM use electron beams and have much higher resolution. 3. Newer high-resolution techniques like confocal and scanning probe microscopy can image individual atoms by using laser beams or atomic interactions between probes and specimens. Specimen preparation and staining remain important for visualizing microscopic structures across different microscopy techniques.

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Louella Artates
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88 views50 pages

The Study of Microbial Structure: Microscopy and Specimen Preparation

1. Microscopy techniques allow observation of microscopic structures through use of lenses and light or electron beams. Specimen preparation through staining and fixing is required to visualize microscopic structures. 2. Various microscopes like bright-field, dark-field, phase-contrast, and fluorescence microscopes use different optical configurations and light sources to visualize specimens. Electron microscopes like TEM and SEM use electron beams and have much higher resolution. 3. Newer high-resolution techniques like confocal and scanning probe microscopy can image individual atoms by using laser beams or atomic interactions between probes and specimens. Specimen preparation and staining remain important for visualizing microscopic structures across different microscopy techniques.

Uploaded by

Louella Artates
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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1

THE STUDY OF MICROBIAL


STRUCTURE: MICROSCOPY
AND SPECIMEN PREPARATION

Scale

Discovery of
Microorganisms
Antony

van
Leeuwenhoe
k (16321723)
first

person
to observe
and describe
microorganisms
accurately

Figure 1.1b

Lenses and the Bending of


Light

light

is refracted (bent) when passing


from one medium to another

refractive

index

measure of how greatly a substance


slows the velocity of light

direction

and magnitude of bending is


determined by the refractive indexes
of the two media forming the interface

Lenses

focus light rays at a specific place called the


focal point

distance between center of lens and focal point


is the focal length

strength of lens related to focal length

short focal length more magnification

Figure 2.2

The Light Microscope

many types

bright-field microscope

dark-field microscope

phase-contrast microscope

fluorescence microscopes

are compound microscopes

image formed by action of 2 lenses

The Bright-Field
Microscope

produces a dark image against a brighter


background

has several objective lenses

parfocal microscopes remain in focus when


objectives are changed

total magnification

product of the magnifications of the ocular lens


and the objective lens

Figure 2.3

10

Figure 2.4

Microscope Resolution

ability of a lens to separate or distinguish small


objects that are close together

wavelength of light used is major factor in


resolution
shorter wavelength greater resolution

11

12

working distance
distance between

the front surface of lens and surface of


cover glass or specimen

13

Figure 2.5

14

Figure 2.6

The Dark-Field
Microscope

produces a bright image of the object against a


dark background

used to observe living, unstained preparations

15

16

Figure 2.7b

The Phase-Contrast
Microscope

17

enhances the contrast between intracellular structures


having slight differences in refractive index

excellent way to observe living cells

18

Figure 2.9

19

Figure 2.10

20

The Differential Interference


Contrast Microscope

creates image by detecting differences in refractive


indices and thickness of different parts of specimen

excellent way to observe living cells

The Fluorescence
Microscope

exposes specimen to ultraviolet, violet, or blue


light

specimens usually stained with fluorochromes

shows a bright image of the object resulting


from the fluorescent light emitted by the
specimen

21

22

Figure 2.12

23

Figure 2.13c and d

Preparation and Staining


of Specimens

increases visibility of specimen

accentuates specific morphological features

preserves specimens

24

25

Fixation
process

by which internal and external


structures are preserved and fixed in
position
process by which organism is killed
and firmly attached to microscope
slide
heat

fixing

preserves

structures

chemical
protects

overall morphology but not internal

fixing

fine cellular substructure and


morphology of larger, more delicate organisms

Dyes and Simple Staining

26

dyes

make internal and external structures of cell more visible


by increasing contrast with background

have two common features

chromophore groups

chemical groups with conjugated double bonds

give dye its color

ability to bind cells

Dyes and Simple Staining

simple staining

a single staining agent is used

basic dyes are frequently used

dyes with positive charges

e.g., crystal violet

27

Differential Staining

divides microorganisms into groups based on


their staining properties

e.g., Gram stain

e.g., acid-fast stain

28

Gram staining

most widely used differential staining procedure

divides Bacteria into two groups based on


differences in cell wall structure

29

30
primary
stain
mordant

decolorization
counterstain
Figure 2.14

positive
negative

31

Figure 2.15c

Escherichia coli a gram-negative rod

Acid-fast staining

particularly useful for staining members of the


genus Mycobacterium
e.g., Mycobacterium tuberculosis causes tuberculosis
e.g., Mycobacterium leprae causes leprosy

high lipid content in cell walls is responsible for


their staining characteristics

32

Staining Specific
Structures

Negative staining

often used to visualize capsules surrounding bacteria

capsules are colorless against a stained background

33

34

Staining Specific Structures

Spore staining

double staining technique

bacterial endospore is one color and vegetative cell is a


different color

Flagella staining

mordant applied to increase thickness of flagella

Electron Microscopy
beams

of
electrons are
used to produce
images
wavelength of
electron beam is
much shorter
than light,
resulting in much
higher resolution
35

Figure 2.20

The Transmission
Electron Microscope

36

electrons scatter when they pass through thin sections


of a specimen

transmitted electrons (those that do not scatter) are


used to produce image

denser regions in specimen, scatter more electrons and


appear darker

37

EM

Figure 2.23

Specimen Preparation

38

analogous to procedures used for light microscopy

for transmission electron microscopy, specimens must


be cut very thin

specimens are chemically fixed and stained with


electron dense material

Other preparation
methods

shadowing

coating specimen with a thin film of a heavy


metal

freeze-etching

freeze specimen then fracture along lines of


greatest weakness (e.g., membranes)

39

40

Figure 2.25

Ebola

41

Fly head

42

The Scanning Electron


Microscope

43

uses electrons reflected from the surface of a specimen


to create image

produces a 3-dimensional image of specimens surface


features

44

Figure 2.27

Newer Techniques in
Microscopy
confocal

microscopy and
scanning probe
microscopy
have extremely
high resolution
can be used to
observe
individual atoms
45

Figure 2.20

Confocal Microscopy

confocal scanning laser microscope

laser beam used to illuminate spots on


specimen

computer compiles images created from each


point to generate a 3-dimensional image

46

47

Figure 2.29

48

Figure 2.30

49

Scanning Probe Microscopy

scanning tunneling microscope

steady current (tunneling current) maintained between


microscope probe and specimen

up and down movement of probe as it maintains current is


detected and used to create image of surface of specimen

50

Scanning Probe Microscopy

atomic force microscope

sharp probe moves over surface of specimen at constant


distance

up and down movement of probe as it maintains constant


distance is detected and used to create image

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